Magnetically Guided Adeno-Associated Virus Delivery for the Spatially Targeted Transduction of Retina in Eyes.

Adeno-associated viruses (AAVs) are intensively explored for gene therapies in general and have found promising applications for treating retina diseases. However, controlling the specificity (tropism) and delivery of AAVs to selected layers, cell types, and areas of the retina is a major challenge to further develop retinal gene therapies. Magnetic nanoparticles (MNPs) provide effective delivery platforms to magnetically guide therapeutics to target cells. Yet, how MNPs can deliver AAVs to transfect particular retina layers and cells remains elusive. Here, we demonstrate that MNPs can be used to transport different AAVs through the retina and to modulate the selective transduction of specific retinal layers or photoreceptor cells in ex vivo porcine explants and whole eyes. Thereby, transduction is triggered by bringing the viruses in close proximity to the target cell layer and by controlling their interaction time. We show that this magnetically guided approach to transport AAVs to selected areas and layers of the retina does not require the cell-specific optimization of the AAV tropism. We anticipate that the new approach to control the delivery of AAVs and to selectively transduce cellular systems can be applied to many other tissues or organs to selectively deliver genes of interest. This article is protected by copyright. All rights reserved.

Prevalence of Psychotropic Medication Use and Psychotropic Polypharmacy in Autistic Adults With or Without Intellectual Disability.

The aim of this study was to compare the rates of psychotropic medication use and psychotropic polypharmacy between autistic adults with and without intellectual disability (ID) and to examine factors associated with psychotropic medication use and psychotropic polypharmacy in autistic adults, stratified by the presence of ID. We conducted a retrospective medical chart review of outpatients with an autism diagnosis aged 18 years and older. The rates of psychotropic medication use and psychotropic polypharmacy were compared between autistic adults with and without ID. Subsequently, logistic regression analyses were performed to identify factors associated with psychotropic medication use and psychotropic polypharmacy in autistic adults with ID and those without ID, respectively. The rates of prevalence of psychotropic medication use and polypharmacy were significantly higher in participants with ID than those without ID (78.6% vs. 58.8% and 49.3% vs. 31.2%; p-values < 0.05). Age, gender, race, residence, presence of mood disorders, presence of schizophrenia, absence of anxiety disorder, number of psychiatric comorbidities, and presence of behaviors that challenge were significantly associated with these outcomes, depending on the presence/absence of ID. The need to optimize pharmacotherapy in autistic adults, stratifying by the presence of ID, is highlighted.

[Clinical Application Examples of a Next-Generation Sequencing based Multi-Genepanel Analysis]. / Klinische Anwendungsbeispiele einer Next-Generation-Sequencing-basierten Multi-Genpanel-Analyse.

This review provides an overview of clinically useful applications of a next-generation sequencing (NGS)-based multi-gene panel testing strategy in the areas of oncology, hereditary tumor syndromes, and hematology. In the case of solid tumors (e.g. lung carcinoma, colon-rectal carcinoma), the detection of somatic mutations contributes not only to a better diagnostic but also therapeutic stratification of those affected. The increasing genetic complexity of hereditary tumor syndromes (e.g. breast and ovarian carcinoma, lynch syndrome/polyposis) requires a multi-gene panel analysis of germline mutations in affected families. Another useful indication for a multi-gene panel diagnostics and prognosis assessment are acute and chronic myeloid diseases. The criteria of the WHO-classification and the European LeukemiaNet-prognosis system for acute myeloid leukemia can only be met by a multi-gene panel test strategy.

Monitoring the antibiotic darobactin modulating the ß-barrel assembly factor BamA.

The ß-barrel assembly machinery (BAM) complex is an essential component of Escherichia coli that inserts and folds outer membrane proteins (OMPs). The natural antibiotic compound darobactin inhibits BamA, the central unit of BAM. Here, we employ dynamic single-molecule force spectroscopy (SMFS) to better understand the structure-function relationship of BamA and its inhibition by darobactin. The five N-terminal polypeptide transport (POTRA) domains show low mechanical, kinetic, and energetic stabilities. In contrast, the structural region linking the POTRA domains to the transmembrane ß-barrel exposes the highest mechanical stiffness and lowest kinetic stability within BamA, thus indicating a mechano-functional role. Within the ß-barrel, the four N-terminal ß-hairpins H1-H4 expose the highest mechanical stabilities and stiffnesses, while the four C-terminal ß-hairpins H5-H6 show lower stabilities and higher flexibilities. This asymmetry within the ß-barrel suggests that substrates funneling into the lateral gate formed by ß-hairpins H1 and H8 can force the flexible C-terminal ß-hairpins to change conformations.

Monitoring the binding and insertion of a single transmembrane protein by an insertase.

Cells employ highly conserved families of insertases and translocases to insert and fold proteins into membranes. How insertases insert and fold membrane proteins is not fully known. To investigate how the bacterial insertase YidC facilitates this process, we here combine single-molecule force spectroscopy and fluorescence spectroscopy approaches, and molecular dynamics simulations. We observe that within 2 ms, the cytoplasmic α-helical hairpin of YidC binds the polypeptide of the membrane protein Pf3 at high conformational variability and kinetic stability. Within 52 ms, YidC strengthens its binding to the substrate and uses the cytoplasmic α-helical hairpin domain and hydrophilic groove to transfer Pf3 to the membrane-inserted, folded state. In this inserted state, Pf3 exposes low conformational variability such as typical for transmembrane α-helical proteins. The presence of YidC homologues in all domains of life gives our mechanistic insight into insertase-mediated membrane protein binding and insertion general relevance for membrane protein biogenesis.

The Safety and Efficacy of Microbial Ecosystem Therapeutic-2 in People With Major Depression: Protocol for a Phase 2, Double-Blind, Placebo-Controlled Study.

BACKGROUND: The gut-brain axis is a bidirectional signaling pathway between the gastrointestinal tract and the brain; it is being studied because of its potential influence in mediating mood, anxiety, and other neuropsychiatric symptoms. Previous research examining the effects of gut microbiota on neuropsychiatric disorders suggests that gut repopulation treatments such as probiotics, microbe therapy, and fecal microbiota transplantation show promising results in treating symptoms of anxiety and depression. This study explores the use of an alternative gut repopulation treatment to fecal microbiota transplantation, known as Microbial Ecosystem Therapeutic (MET)-2, as an intervention against symptoms of depression. MET-2 is a daily, orally administered capsule containing 40 bacterial strains purified from a single healthy donor. OBJECTIVE: The primary aim of this study is to assess changes in mood in people with major depression that occur pre-, post-, and during the administration of MET-2. The secondary aims are to assess changes in anxiety symptoms, blood biomarker concentrations, and the level of repopulation of healthy gut bacteria as a response to treatment. METHODS: In this study, we will recruit 60 adults aged between 18 and 45 years old with major depression and randomly assign them to treatment or placebo groups. Patients in the treatment group will receive MET-2 once a day for 6 weeks, whereas patients in the placebo group will receive a matching placebo for 6 weeks. Participants will complete biweekly visits during the treatment period and a follow-up visit at 2 weeks post treatment. As a primary outcome measure, participants' mood will be assessed using the Montgomery-Asberg Depression Rating Scale. Secondary outcome measures include changes in mood, anxiety, early stress, gastrointestinal symptoms, and tolerability of MET-2 treatment using a series of clinical scales and changes in blood markers, particularly immunoglobulins (Igs; IgA, IgG, and IgM) and inflammatory markers (C-reactive protein, tumor necrosis factor-α, transforming growth factor-ß, interleukin-6, and interleukin-10). Changes in the relative abundance, diversity, and level of engraftment in fecal samples will be assessed using 16S rRNA sequencing. All data will be integrated to identify biomarkers that could indicate disease state or predict improvement in depressive symptoms in response to MET-2 treatment. RESULTS: Given the association between the gut microbiome and depression, we hypothesized that participants receiving MET-2 would experience greater improvement in depressive symptoms than those receiving placebo owing to the recolonization of the gut microbiome with healthy bacteria modulating the gut-brain axis connection. CONCLUSIONS: This study is the first of its kind to evaluate the safety and efficacy of a microbial therapy such as MET-2 in comparison with placebo for major depressive disorder. We hope that this study will also reveal the potential capabilities of microbial therapies to treat other psychiatric illnesses and mood disorders. TRIAL REGISTRATION: ClinicalTrials.gov NCT04602715; https://clinicaltrials.gov/ct2/show/NCT04602715. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/31439.

Contributions of cholinergic receptor muscarinic 1 and CYP1A2 gene variants on the effects of plasma ratio of clozapine/N-desmethylclozapine on working memory in schizophrenia.

BACKGROUND: Clozapine has heterogenous efficacy in enhancing working memory in schizophrenia. We have previously hypothesized that this is due to opposing effects of clozapine and its metabolite, N-desmethylclozapine, at the muscarinic M1 receptor and demonstrated that a lower clozapine/N-desmethylclozapine ratio is associated with better working memory than clozapine or N-desmethylclozapine levels alone. AIMS: In this study, we expanded the above hypothesis to explore whether genetic variation in the cholinergic receptor muscarinic 1 gene, encoding the M1 receptor, affects the relationship between clozapine/N-desmethylclozapine and working memory. Further, we explored whether CYP1A2 gene variants affect the ratio of clozapine/N-desmethylclozapine and by this, working memory performance. METHODS: We evaluated two functionally significant single nucleotide polymorphisms, rs1942499 and rs2075748, in cholinergic receptor muscarinic 1, with the haplotype T-A associated with lower transcriptional activity than the haplotype C-G. Further, we examined CYP1A2 *1F, with *1F/*1F conferring high inducibility in the presence of smoking. RESULTS: In a sample of 30 patients with schizophrenia on clozapine monotherapy, clozapine/N-desmethylclozapine was correlated with working memory only in non-carriers of the haplotype T-A of the cholinergic receptor muscarinic 1 gene. Interaction of CYP1A2 genotype and smoking status significantly affected clozapine concentrations, but there were no significant effects of CYP1A2 genotype and smoking status on the relationship between clozapine/N-desmethylclozapine on working memory. CONCLUSIONS: Our finding that the relationship between clozapine/N-desmethylclozapine and working memory is specific to patients with potentially higher transcription of M1 receptor (i.e. non-carriers of the haplotype T-A of cholinergic receptor muscarinic 1) supports a cholinergic mechanism underlying this relationship.

Pharmacogenomic Studies in Intellectual Disabilities and Autism Spectrum Disorder: A Systematic Review.

BACKGROUND: Individuals with intellectual disability (ID) and autism spectrum disorder (ASD) often receive psychotropic medications such as antipsychotics and antidepressants to treat aberrant behaviors and mood symptoms, frequently resulting in polypharmacy and drug-related adverse effects. Pharmacogenomic (PGx) studies with ASD and/or ID (ASD/ID) have been scarce despite the promise of optimizing treatment outcomes. We reviewed the literature on PGx studies with antipsychotics and antidepressants (e.g., treatment response and adverse effects) in ASD/ID. METHODS: We performed a systematic review using MEDLINE, Embase, and PsycINFO, including peer-reviewed original articles in English referring to PGx in the treatment of ASD/ID in any age groups (e.g., treatment response and adverse effects). RESULTS: A total of 28 PGx studies using mostly candidate gene approaches were identified across age groups. Notably, only 3 studies included adults with ASD/ID while the other 25 studies focused specifically on children/adolescents with ASD/ID. Twelve studies primarily investigated treatment response, of which 5 and 6 studies included patients treated with antipsychotics and antidepressants, respectively. Most interesting results for response were reported for 2 sets of candidate gene studies, namely: (1) The DRD3 Ser9Gly (rs6280) polymorphism was examined in patients treated with risperidone in 3 studies, 2 of which reported an association with risperidone treatment response and (2) the SLC6A4 5-HTTLPR polymorphism and treatment response to antidepressants which was investigated in 4 studies, 3 of which reported significant associations. In regard to side effects, 9 of 15 studies focused on hyperprolactinemia in patients treated with risperidone. Among them, 7 and 5 studies examined the impact of CYP2D6 and DRD2 Taq1A polymorphisms, respectively, yielding mostly negative study findings. CONCLUSIONS: There is limited data available on PGx in individuals with ASD/ID and in particular in adults. Given the potential for PGx testing in improving treatment outcomes, additional PGx studies for psychotropic treatment in ASD/ID across age groups are warranted.

Regulation of melanocortin-4-receptor (MC4R) expression by SNP rs17066842 is dependent on glucose concentration.

Melanocortin-4-receptor (MC4R) gene codes for a G-protein-coupled receptor that is highly expressed in the hypothalamus and involved in the regulation of appetite. Single-nucleotide polymorphisms (SNPs) in the MC4R gene region have been associated with obesity, type 2-diabetes (T2D) and with antipsychotic-induced weight gain. Of these, rs17066842 (G>A) in the MC4R promoter region is the top variant associated with obesity and diabetes. In this study, we investigated the effect of rs17066842 on MC4R expression at various glucose concentrations using reporter gene expression in the SH-SY5Y cell line and regulation of MC4R expression in human cerebral organoids. We observed that higher glucose concentrations significantly reduced MC4R mRNA expression in SH-SY5Y cells. In addition, at high glucose concentrations, the luciferase reporter plasmid containing the MC4R promoter insert with the G-allele of rs170066842 showed significantly reduced activity compared to the A-allele carrying plasmid. The immediate early gene product, early growth-response 1 (EGR-1), was identified to bind to the sequence containing the G-allele at rs17066842 but not to the A-allele-containing sequence. Interestingly, in human induced pluripotent stem cell (hiPSC)-derived cerebral organoids, we observed increased MC4R expression in response to high glucose exposure. These opposite observations might suggest that glucose regulation is complex and may be cell-specific. This study provides evidence that rs17066842 regulates MC4R gene expression through binding of EGR-1 and that this process is influenced by glucose concentration.

Investigation of the Gut Microbiome in Patients with Schizophrenia and Clozapine-Induced Weight Gain: Protocol and Clinical Characteristics of First Patient Cohorts.

BACKGROUND: Emerging evidence suggests an important role of the human gut microbiome in psychiatry and neurodevelopmental disorders. An increasing body of literature based on animal studies has reported that the gut microbiome influences brain development and behavior by interacting with the gut-brain axis. Furthermore, as the gut microbiome has an important role in metabolism and is known to interact with pharmaceuticals, recent evidence suggests a role for the microbiome in antipsychotic-induced metabolic side effects in animals and humans. PURPOSE: Here we present the protocol for a two-phase study investigating the gut microbiome in healthy controls and in patients with schizophrenia treated with antipsychotics. METHODS: Phase I of our study involves humans exclusively. We recruit 25 patients who are chronically treated with clozapine and compare them with 25 healthy controls matched for age, sex, BMI, and smoking status. A second cohort consists of 25 patients newly starting on clozapine, and a third cohort includes 25 antipsychotic-naive patients. The patients in the second cohort and third cohort are prospectively assessed for up to 6 and 12 weeks, respectively. Phase II of this study will incorporate microbiota humanized mouse models to examine the influence of human fecal transplant on metabolic parameters and the gut-brain axis. Progress and Future Directions: We are underway with the first participants enrolled in all phase I treatment cohorts. This study will contribute to elucidating the role of the gut microbiome in schizophrenia and metabolic side effects. In addition, its results may help to explore potential therapeutic targets for antipsychotic-induced metabolic side effects.

Magnetically guided virus stamping for the targeted infection of single cells or groups of cells.

To understand and control complex tissues, the ability to genetically manipulate single cells is required. However, current delivery methods for the genetic engineering of single cells, including viral transduction, suffer from limitations that restrict their application. Here we present a protocol that describes a versatile technique that can be used for the targeted viral infection of single cells or small groups of cells in any tissue that is optically accessible. First, cells of interest are selected using optical microscopy. Second, a micropipette-loaded with magnetic nanoparticles to which viral particles are bound-is brought into proximity of the cell of interest, and a magnetic field is applied to guide the viral nanoparticles into cellular contact, leading to transduction. The protocol, exemplified here by stamping cultured neurons with adeno-associated viruses (AAVs), is completed in a few minutes and allows stable transgene expression within a few days, at success rates that approach 80%. We outline how this strategy is applied to single-cell infection in complex tissues, and is feasible both in organoids and in vivo.

Predicting Worsening Suicidal Ideation With Clinical Features and Peripheral Expression of Messenger RNA and MicroRNA During Antidepressant Treatment.

OBJECTIVE: To investigate how the combination of clinical and molecular biomarkers can predict worsening of suicidal ideation during antidepressant treatment. METHODS: Samples were obtained from 237 patients with major depressive disorder (DSM-IV criteria) treated with either duloxetine or placebo in an 8-week randomized controlled trial. Data were collected between 2007 and 2011. The relationship between treatment-worsening suicidal ideation (TWSI) and a number of clinical variables, as well as peripheral expression of messenger RNA (mRNA) and microRNA (miRNA), was assessed at baseline. We generated 4 predictive models for TWSI: clinical, mRNA, miRNA, and a combined model comprising the best predictive variables from clinical, mRNA, and miRNA data. RESULTS: Eleven patients (9.8%) presented with TWSI in the duloxetine group. Among the clinical variables, only baseline depressive severity was found to be mildly predictive of TWSI. Two mRNAs (stathmin 1 [STMN1] and protein phosphatase 1 regulatory subunit 9B [PPP1R9B]) and 2 miRNAs (miR-3688 and miR-5695) were identified that were significantly predictive of TWSI when mRNA and miRNA were assessed separately (P = .002, .044, .004, and .005, respectively). The best model included baseline depression severity and expression of STMN1 and miR-5695 and predicted TWSI with area under the curve = 0.94 (P < .001). Additionally, the combined model did not significantly predict TWSI in the placebo group. CONCLUSIONS: This study generated a predictive tool for TWSI that combines both biological and clinical variables. These biological variables can be easily quantified in peripheral tissues, thus rendering them viable targets to be used in both clinical practice and future studies of suicidal behaviors. TRIAL REGISTRATION: ClinicalTrials.gov identifiers: NCT00635219, NCT00599911, and NCT01140906.

Insertion and folding pathways of single membrane proteins guided by translocases and insertases.

Biogenesis in prokaryotes and eukaryotes requires the insertion of α-helical proteins into cellular membranes for which they use universally conserved cellular machineries. In bacterial inner membranes, insertion is facilitated by YidC insertase and SecYEG translocon working individually or cooperatively. How insertase and translocon fold a polypeptide into the native protein in the membrane is largely unknown. We apply single-molecule force spectroscopy assays to investigate the insertion and folding process of single lactose permease (LacY) precursors assisted by YidC and SecYEG. Both YidC and SecYEG initiate folding of the completely unfolded polypeptide by inserting a single structural segment. YidC then inserts the remaining segments in random order, whereas SecYEG inserts them sequentially. Each type of insertion process proceeds until LacY folding is complete. When YidC and SecYEG cooperate, the folding pathway of the membrane protein is dominated by the translocase. We propose that both of the fundamentally different pathways along which YidC and SecYEG insert and fold a polypeptide are essential components of membrane protein biogenesis.

Genetic study of neuregulin 1 and receptor tyrosine-protein kinase erbB-4 in tardive dyskinesia.

OBJECTIVES: Tardive dyskinesia (TD) is a movement disorder that may develop as a side effect of antipsychotic medication. The aetiology underlying TD is unclear, but a number of mechanisms have been proposed. METHODS: We investigated single-nucleotide polymorphisms (SNPs) in the genes coding for neuregulin-1 and erbB-4 receptor in our sample of 153 European schizophrenia patients for possible association with TD. RESULTS: We found the ERBB4 rs839523 CC genotype to be associated with risk for TD occurrence and increased severity as measured by the Abnormal Involuntary Movement Scale (AIMS) (P = .003). CONCLUSIONS: This study supports a role for the neuregulin signalling pathway in TD, although independent replications are warranted.

Vaccinia virus hijacks EGFR signalling to enhance virus spread through rapid and directed infected cell motility.

Cell motility is essential for viral dissemination1. Vaccinia virus (VACV), a close relative of smallpox virus, is thought to exploit cell motility as a means to enhance the spread of infection1. A single viral protein, F11L, contributes to this by blocking RhoA signalling to facilitate cell retraction2. However, F11L alone is not sufficient for VACV-induced cell motility, indicating that additional viral factors must be involved. Here, we show that the VACV epidermal growth factor homologue, VGF, promotes infected cell motility and the spread of viral infection. We found that VGF secreted from early infected cells is cleaved by ADAM10, after which it acts largely in a paracrine manner to direct cell motility at the leading edge of infection. Real-time tracking of cells infected in the presence of EGFR, MAPK, FAK and ADAM10 inhibitors or with VGF-deleted and F11-deleted viruses revealed defects in radial velocity and directional migration efficiency, leading to impaired cell-to-cell spread of infection. Furthermore, intravital imaging showed that virus spread and lesion formation are attenuated in the absence of VGF. Our results demonstrate how poxviruses hijack epidermal growth factor receptor-induced cell motility to promote rapid and efficient spread of infection in vitro and in vivo.

Pharmacogenetic guidelines and decision support tools for depression treatment: application to late-life.

Late-life depression (LLD) is a major depressive disorder that affects someone after the age of 60 years. LLD is frequently associated with inadequate response and remission from antidepressants, in addition to polypharmacy. Pharmacogenetics offers a promising approach to improve clinical outcomes in LLD via new discoveries determining the genetic basis of response rates and side effects, as well as the development of tailored pharmacogenetic-based decision support tools. This invited review evaluates the LLD pharmacogenetic evidence base and the extent to which this was incorporated into existing commercial decision support tools and clinical pharmacogenetic guidelines.

Single-Molecule Force Spectroscopy of Transmembrane ß-Barrel Proteins.

Single-molecule force spectroscopy (SMFS) has been widely applied to study the mechanical unfolding and folding of transmembrane proteins. Here, we review the recent progress in characterizing bacterial and human transmembrane ß-barrel proteins by SMFS. First, we describe the mechanical unfolding of transmembrane ß-barrels, which follows a general mechanism dictated by the sequential unfolding and extraction of individual ß-strands and ß-hairpins from membranes. Upon force relaxation, the unfolded polypeptide can insert stepwise into the membrane as single ß-strands or ß-hairpins to fold as the native ß-barrel. The refolding can be followed at a high spatial and temporal resolution, showing that small ß-barrels are able to fold without assistance, whereas large and complex ß-barrels require chaperone cofactors. Applied in the dynamic mode, SMFS can quantify the kinetic and mechanical properties of single ß-hairpins and reveal complementary insight into the membrane protein structure and function relationship. We further outline the challenges that SMFS experiments must overcome for a comprehensive understanding of the folding and function of transmembrane ß-barrel proteins.

Mechanism of membrane pore formation by human gasdermin-D.

Gasdermin-D (GSDMD), a member of the gasdermin protein family, mediates pyroptosis in human and murine cells. Cleaved by inflammatory caspases, GSDMD inserts its N-terminal domain (GSDMDNterm) into cellular membranes and assembles large oligomeric complexes permeabilizing the membrane. So far, the mechanisms of GSDMDNterm insertion, oligomerization, and pore formation are poorly understood. Here, we apply high-resolution (≤ 2 nm) atomic force microscopy (AFM) to describe how GSDMDNterm inserts and assembles in membranes. We observe GSDMDNterm inserting into a variety of lipid compositions, among which phosphatidylinositide (PI(4,5)P2) increases and cholesterol reduces insertion. Once inserted, GSDMDNterm assembles arc-, slit-, and ring-shaped oligomers, each of which being able to form transmembrane pores. This assembly and pore formation process is independent on whether GSDMD has been cleaved by caspase-1, caspase-4, or caspase-5. Using time-lapse AFM, we monitor how GSDMDNterm assembles into arc-shaped oligomers that can transform into larger slit-shaped and finally into stable ring-shaped oligomers. Our observations translate into a mechanistic model of GSDMDNterm transmembrane pore assembly, which is likely shared within the gasdermin protein family.

POTRA Domains, Extracellular Lid, and Membrane Composition Modulate the Conformational Stability of the ß Barrel Assembly Factor BamA.

The core component BamA of the ß barrel assembly machinery (BAM) adopts several conformations, which are thought to facilitate the insertion and folding of ß barrel proteins into the bacterial outer membrane. Which factors alter the stability of these conformations remains to be quantified. Here, we apply single-molecule force spectroscopy to characterize the mechanical properties of BamA from Escherichia coli. In contrast to the N-terminal periplasmic polypeptide-transport-associated (POTRA) domains, the C-terminal transmembrane ß barrel domain of BamA is mechanically much more stable. Exposed to mechanical stress this ß barrel stepwise unfolds ß hairpins until unfolding has been completed. Thereby, the mechanical stabilities of ß barrel and ß hairpins are modulated by the POTRA domains, the membrane composition and the extracellular lid closing the ß barrel. We anticipate that these differences in stability, which are caused by factors contributing to BAM function, promote conformations of the BamA ß barrel required to insert and fold outer membrane proteins.

Virus stamping for targeted single-cell infection in vitro and in vivo.

Genetic engineering by viral infection of single cells is useful to study complex systems such as the brain. However, available methods for infecting single cells have drawbacks that limit their applications. Here we describe 'virus stamping', in which viruses are reversibly bound to a delivery vehicle-a functionalized glass pipette tip or magnetic nanoparticles in a pipette-that is brought into physical contact with the target cell on a surface or in tissue, using mechanical or magnetic forces. Different single cells in the same tissue can be infected with different viruses and an individual cell can be simultaneously infected with different viruses. We use rabies, lenti, herpes simplex, and adeno-associated viruses to drive expression of fluorescent markers or a calcium indicator in target cells in cell culture, mouse retina, human brain organoid, and the brains of live mice. Virus stamping provides a versatile solution for targeted single-cell infection of diverse cell types, both in vitro and in vivo.